The re-programming of post-natal somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of stem cell specifying transcription factors has many exciting potential applications for improving human health. iPSCs were initially developed in the murine model, and just a few years later, human iPS cells were created. However, there are numerous hurdles to moving iPSC forward into clinical regenerative medicine applications. First and most important are safety concerns, most seriously the consequences of administering primitive pluripotent cells that may have the potential to form tumors, if differentiation is incomplete or inefficient. Second, there are significant challenges to the efficient differentiation of iPSCs into functional adult tissues. Protocols for differentiation of iPSCs towards even well-characterized hematopoietic stem cells are inefficient, inconsistent and result in aberrant or embryonic hematopoiesis. Design of methods for direct delivery or facilitation of homing of iPSCs or their progeny to appropriate locations in the body will also be a major challenge. While murine models are invaluable tools, it will be critical to develop more relevant models for clinical development of iPSCs. Human iPSCs can be implanted in immunodeficient mouse strains and form teratomas, but the next steps in development, requiring functional differentiation and appropriate delivery or homing, may be impossible to model in xenografts. Scale-up of laboratory procedures developed in mice to human therapies would also be very difficult to develop solely using murine-murine or human-murine xenograft models. The rhesus macaque non-human primate (NHP) model will be a valuable resource to clear hurdles preventing clinical development. Teratoma formation and other safety issues can be directly assessed utilizing autologous rhesus iPSCs. Differentiation, homing and other parameters critical for efficacy can be modeled. Tissue damage models such as pancreatic beta cell or hematopoietic stem cell ablation are well established in macaques. Development of rhesus iPSCs at the NIH takes advantage of our unique expertise in NHP transplantation and in the development of novel cell and gene therapies in this valuable model. Thsi project began last year, and we have already published a paper taking advantage of our expertise in vector integration site retrieval and analysis to demonstrate that in human iPSCs, vector integration sites do not appear to play a role in promoting successful reprogramming of iPSCs. This is reassuring for at least preclinical and model development, allowing continued use of lentiviral vectors for reprogramming, given their much greater efficiency compared to non-integrating vectors. We have now optimized conditions to derive rhesus iPSCs, and have successfully shown that rhesus iPSCs can be created from rhesus marrow mesenchymal cells, skin fibroblasts, or CD34+ cells, utilizing either retroviral or lentiviral vectors. The conditions utilized for murine and human ESCs and iPSCs were not successful using rhesus cells, and we have developed new conditons, based on the optimal conditions for growing rhesus ESCs.We have derived multiple rhesus iPSC clones utilizing a polycistronic lentiviral vector that then allows cre-mediated excision of the pluripotency factor cassette, leaving only a small DNA tag that can be utilized to identify and track iPS-derived cells in vivo. These clones are pluripotent as assayed in a murine teratoma assay, express all pluripotency markers, and can be differentiated to endodermal, mesodermal and ectodermal cell types. Our rhesus iPSCs express alkaline phosphatase, shut off the reprogramming vectors and morphologically resemble rhesus ESCs. For in vivo studies in autologous rhesus, excision of the potentially-immunogenic reprogramming factors is likely required, so we have now created rhesus iPSCs with an exicisable reprogramming cassette, and shown all retained functions following cre-mediated excision. We have begun stepwise development of a rhesus autologous teratoma model which we believe is critical for further development and safety testing of iPSCs, and far more relevant than the xenograft models that have been utilized previously. Our initial experiments did not show autologous teratoma formation, despite concurrent studies showing teratoma formation with the same cells in immunodeficient mice. We are stepwise removing all possible xenogeneic proteins from iPSC culture, adapting cells to feeder-free, Matrigel free, and eventually serum-free conditions, all which will likely be required for transplantion of iPSC cells or their progeny in vivo in normal animals. We have developed approaches for genetic modification of rhesus iPSCs and have introduced the inducible caspase 9 suicide gene into these cells and demonstrated effective killing of iPSCs in vitro and in vivo, following exposure to the dimerizer AP1903.